The goal of this project is to develop the next generation of targeted T-cells with chimeric antigen receptors (CARs) for use in carcinomas and hematologic malignancies. CARs are now beginning to show activity in a number of pilot clinical trials and they have significant potential for therapy of many cancers that are currently incurable, however two issues have emerged that provide a barrier to rapid progress in the field: 1) available preclinical models have not accurately predicted the safety of CARs, and unexpected toxicities from cytokine release and tissue damage have been reported in recent trials; 2) high costs and long lead times required for vector production have slowed the clinical application of T cells expressing CARs, and prevent a facile and iterative approach to optimize CAR design and determine the optimal target structures on tumor cells. This is currently a major problem to the field, as our preliminary data has conclusively demonstrated that CARs have potent activity in a pilot clinical trial. Our preliminary data further establishes that T lymphocytes can be efficiently modified by mRNA electroporation without integration-associated safety concerns, and that infusions of RNA CAR T cells mediate robust antitumor effects in preclinical humanized models with disseminated tumor xenografts. Thus, this new platform affords the possibility of rapidly testing potent RNA CARs for antitumor activity, and in the event of toxicity, limiting off-target exposure by discontinuing CAR administration. Because late relapses due to tumor escape variants in pre-clinical models have been identified, it will be important to test combinations of CAR T cells to augment antitumor effects and prevent recurrence. The theme of the project remains essentially the same from the previous grant and our new specific aims are designed to: (1) enhance the delivery and trafficking of CARs using mRNA electroporated T cells; (2) optimize the cell used for CAR therapy and understand the mechanisms that lead to enhanced persistence; and (3) define whether combination CARs improve antitumor activity and decrease the emergence of resistant tumor. In summary, these overlapping studies will test the central hypothesis that multiple CARs (i.e., a CAR fleet) will improve CAR immunotherapy compared to therapy with monoclonal CARs. Furthermore, these studies will establish the safety and feasibility of increasing the therapeutic index of T cells engineered to express powerful activation domains without the associated safety concerns of integrating viral vectors.